R,,-4. GRE.\THOUSE, H. G. SHIRK. X.X. SCHWARTZ A N D MILTONHARRIS Vol. 76 1658 F. U'. ~ I I N OG separated almost a t once. These were collected and dried, giving 0.30 g. (27%) of colorless needles, m.p. 156-160°. The crystals (0.30 g.) were recrystallized from 12 ml. of cyclohexane-benzene ( 5 : l ) , giving 0.25 g. of colorless needle clusters, m.p. 158-159.5'. For analysis a 150-mg. sample was recrystallized from about 7 ml. of absolute ethanol, m.p. 159-160O. Anal. Calcd. for CI3Hl8BrK206 (373.17): C, 41.84; H, 3.51; Br, 21.42; N, 7.51, Found: C, 31.83; H, 3.66; Br, 21.50; N,7.51. The compound gave a positive Beilstein halogen test, but was inert to alcoholic silver nitrate even after one minute boiling. The compound w.aq riot attacked on prolonged stirring with l j AI aqueous amnionin or 1 d f aqueous sodium hydroxide a t 2.5'. I t wn1 attacked by boiling sodium hydroxide, giving a red solution. \Vhen heated with strong ethanolic ammonia in a sealed tube for several hours a t looo, the compound gradually dissolved, giving a solid product which we have not yet been able to purify.
solution was again filtered, and evaporated down to an oily residue. The original residue (consisting mainly of silver iodide) was washed with 20 ml. of chloroform. The chloroform solution was used t o dissolve the above oily residue. The resulting solution was concentrated t o about 10 ml. and 10 ml. of cyclohexane was added at the b.p. On cooling, 218 mg. of crystals, m.p. 160-188", was obtained. The product was recrystallized from acetic acid, giving 52 mg. (10%) of the diol bis-dinitro-te, yellow prisms, m.p. 181-182', reported'8 m.p. 179 . (The corresponding iodohydrin ester reportedlygbmelts a t 159O.) DL-trans-Cyclohexanediol-l,2 Dibenzoate.-The procedure was similar to that for the bis-dinitrobenzoate. From 1 1 millimoles of cyclohexene, 10 millimoles of iodine and 21 millimoles of silver benzoate (reflux period 3 hours) there \vas obtained an oily, yellowish crude product. This recrystallized from nbyolute ethanol, giving 1.4 g. (44%) of the diol dibenzoate as colorless prisms, m.p. 92.,5-93..5" (block), reported'* m.p. 93'.
DL-trans-Cyclohexanediol-l,Z Bis-3,5-dinitrobenzoate.-
Acknowledgment.-\\-e are indebted to Tennyson Campbell and Daniel Glick for assistance i n some of the preparative work. Financial support by the Xational Research Council, the Research Council of Ontario and the Advisory Committee on Scientific Research is gratefully acknowledged.
T o 893 mg. (2.8 millimoles) of dry, finely pulverized silver 3,5-dinitrobenzoate in 3.0 ml. of dry benzene was added dropwise 254 mg. ( 1.0 millimole) of iodine in 2 . 5 ml. of benzene. T o the resulting light tan suspension w:is xdded dropwise 0.112 ml. (90.3 mg., 1.1 millimoles) of cylohexene in 1.2 ml. of benzene. After a few minutes stirring, the mixture was boiled under reflux for one hour. The hot mixture was filtered at once. On cooling, the hot filtrate gave a small amount of crystals, presurn.~bly of the complex AgI(CBHaCO0 12. The
[CONTRIBUTION FROM
THE
(18) N. A. B. Wilson and J. Reed, J . Chem. Soc., 1269 (19:i5),
TORONTO, CANADA
HARRIS RESEARCH LABORATORIES]
Biosynthesis of C14-Specifically Labeled Cellulose by Accfobacfer xylinurn. I. From D-Glucose-l-C14 with and without Ethanol' BY
FRANCIS
\v. M I N O R ,
GLENNA. GREATHOUSE, HAROLD G. SHIRK, ANTHONYhf. ~ I I L T OHARRIS N
SCHWARTZ AND
RECEIVEDOCTOBER 24, 1953 C14-Specificallylabeled cellulose was biosynthesized by Acetobacter xylinum from ~-glucose-l-C1~ as the sole labeled carbon source. The presence of ethanol in the medium increased the yield and the quantity of C" found in the cellulose. The distribution of CI4 in D-glucose from the bacterial cellulose hydrolysate indicated that approximately 82% of the activity was in position I for the cellulose produced from n-glucose-l-C1' in the ethanol-free medium compared t o 70% ,in that position for the cellulose produced with ethanol present. Positions 3 and 4 contained the remainder of the activity in approximately equal amounts. The cellulose had a lower specific radioactivity than the D-glucose-1-C" that was supplied. This result shows that some of the original hexose units are cleaved prior t o cellulose formation. Thus, polymerization of the D-glucose as such, without prior chain cleavage, is not the sole mechanism of cellulose biosynthesis by this bacterium.
Introduction This study was initiated to determine the ability of Acetobacter xylinum to produce labeled cellulose from various C14-specifically labeled carbohydrates and derivatives used as substrates for the growth of the organism, and a t the same time to provide some information regarding the involved mechanism in cellulose formation.2 It is the purpose of this paper to report experiments concerned with the biosynthesis of C14-specificallylabeled cellulose by this bacterium when cultured in suitable media containing ~-glucose-l-C14. Since the inclusion of ethanol in the culture medium has been observed to increase the yield of cellulose from a given quantity of D-glucose,Yexperiments were performed to determine what advantage, if any, such addition (1) This series of papers is based on work supported by t h e Atomic Energy Commission under contract AT(30-1)-915 with t h e Harris Research Laboratories. ( 2 ) G . A. Greatbou?e, Scitance, 117, 553 (1953). ( 8 ) H. I,. A. 'l'arr and €1. Hibbert, Con. J . Rescorch, 4 , 37%(1930).
might have with respect to the distribution and specific activity of C14 in the cellulose. The biosynthesis of cellulose by A . xylinum a t the surface of appropriate media was first noted by Brown4 in 1886. Corroboration that this membraneous bacterial product, synthesized from either D-ghlcose or other suitable carbohydrate substrates, is chemically identical to cellulose produced by higher plants was obtained by Hibbert and Barsha6-7 employing chemical analytical procedures in conjunction with X-ray techniques. Others*-'o have also compared the X - r i y patterns of bacterial cellulose with those of cotton cellulose. Ifore recently electron microscopic techniques (1) A. J. Brown, J . Chem. Sac,, 49, 172 (1888). ( 5 ) H. Hibbert a n d J . Barsha, Can. J. Research, 6 , 580 (1931). (fi) Ii. Hibbert a n d J. Barsha. THISJOURNAL, 53, 3007 (1931). ( 7 ) J . Rarsha and H. Hibbert, Can. J . Research, 10, 170 (1934). (8) H. 5Iark and G . V. Susich, 2. p k y s i k . Chem., B4,431 (19291, (!I) J. T.'yxert and 1:. I,uft, ibid., B1, 468 (1930). (10) Y Khouvine, C. Champetier and I
